Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Abstract

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts

“…screened reporter libraries with six randomized nucleotides 5′ of UAG and concluded that neither tRNA identity nor amino acid chemical properties influence termination efficiency ( 64 ). Instead they proposed that two 5′ adenines are major determinants of termination efficiency although a recent transcriptome-wide readthrough study in which eRF1 levels were reduced found no such bias ( 65 ). One study using a mouse cell line to monitor readthrough, concluded that neither the identity of the terminal amino acid nor the P-site codon frequency affected termination efficiency ( 66 ).…”

Direct detection of RNA repair by nanopore sequencing

“…Poly(A) selected mRNA libraries were mapped to an S. cerevisiae transcriptome reference 32 , which contains both pre- and post-spliced mRNA references (https://github.com/Jacobson-Lab/yeast_transcriptome_v5). Reads were aligned using Minimap2 33 in a splice-aware fashion with specific parameters (minimap2 -ax splice -uf -k14).…”

Ribosome-bound Upf1 forms distinct 80S complexes and conducts mRNA surveillance

“…RNA-seq and ribosome profiling reads were aligned to a yeast transcriptome (available at ) using bowtie ( Langmead et al 2009 ), and transcript abundance was determined using RSEM ( Li and Dewey 2011 ), as described previously ( Mangkalaphiban et al 2021 ). For intron-containing genes, spliced (“mRNA”) and unspliced (“pre-mRNA”) isoforms were indexed as separate entries.…”

“…Therefore, reads arising from UPF factor transcripts were discarded from these and all subsequent ribosome profiling libraries analyzed. PCR duplicates identified via the 4N barcodes on either side of the read were removed ( Mangkalaphiban et al 2021 ). Read processing statistics for ribosome profiling libraries are provided in Supplemental Table S1 .…”